The present invention generally relates to identifying sequence variations in genes, such as single nucleotide polymorphisms (SNP), and more specifically to using melt curves from polymerase chain reactions (PCR) apparatus to identify the sequence variations.
Real-time PCR is used to detect and quantify target nucleotide sequences. In PCR, one or more reaction wells contain a DNA template that contains the DNA region (target) to be amplified. The temperature of the reaction well is increased so that the DNA dissociates into two single strands. The temperature is then lowered so that primers that are complementary to the area flanking the target sequence then bind. The temperature is then increased slightly to dissociate the single strand and primer bond. The DNA polymerase can then synthesize a new DNA to provide for amplification of the DNA.
The exponential amplification of a sequence is monitored in real time, e.g., by fluorescence. Commonly, a fluorescent dye is used, which only reports the presence of double-stranded DNA. Typically, the dyes do not distinguish sequences and can thus report the amplification of undesired targets. These undesired sequences can be detected during a dissociation step. During dissociation, the doublestranded PCR products melt into single strands, so fluorescence is diminished. Often a melting process is performed after amplification has been fully achieved.
A melt curve can be produced by plotting the loss of fluorescence against a gradual increase in temperature. The detection of different melt curves implies the presence of different sequences. This technique has been used for the detection of single-nucleotide polymorphisms, allelic discrimination, and strain typing of microorganisms.
However, the determination of differences among different melt curves is difficult and may not be repeatable. Therefore, improved methods and systems for detecting sequence variation using melt curves is desirable to provide greater accuracy, reliability, and consistency of the results.